Brain mitochondria are relatively resistant to calcium-induced mitochondrial permeability transition (MPT), with heterogenic response to the insult. The cause for this heterogeneity is not clear, so we studied the distribution of a key regulator of the MPT, cyclophilin D (cypD), within the rat brain by using immunohistology and Western blotting. Motor and parietal cortex, hippocampus, striatum, substantia nigra, ventral tegmental area, septum, and mammillary nucleus displayed a strong immunoreactivity to cypD within specific subpopulation of neurons. The staining was punctate and intense, particularly in perinuclear regions of cells. Apart from neurons, a subpopulation of astrocytes and NG2-positive cells showed higher cypD immunoreactivity. Double staining of cypD with cytochrome oxidase confirmed the mitochondrial specificity of cypD immunoreactivity. The neurons with high levels of cypD also expressed glutamate decarboxylase (GAD) and the calcium binding protein parvalbumin or calbinding D-28k, identifying these cells as interneurons. Western blots confirmed our immunohistochemical findings, showing significantly higher levels of cypD in crude mitochondria of substantia nigra compared with cortex or striatum. Furthermore, nonsynaptic mitochondria representing mainly mitochondria from cell bodies of neurons and glia have about 16% higher levels of cypD compared with synaptic mitochondria that are localized in presynaptic buttons. These data suggest that the underlying factor of heterogenic response of isolated brain mitochondria to MPT-inducing insults can be the different expression levels of cypD, with mitochondria originated from interneurons as the most sensitive. © 2008 Wiley-Liss, Inc.